"We live in a world of color--color is in the trees and sky around us, in our clothes, and in our homes. When the colors of familiar things differ from what we expect, we are usually upset--greenish skies warrant of bad weather, black clothes denote mourning, intense colors in the home stimulate or agitate. The same applies to food. Consumers first judge the quality of the food product by its color." Food Technology, page 49 (July, 1986). However much one may decry a consumer attitude which places higher priority on visual impact than on gustatory and nutritional value, it is an attitude which must be actively confronted in the marketplace.
Although naturally occurring pigments perforce were the first used food colorants, the development of chemistry as a discipline led to many synthetic dyes, especially anilines, to supplant naturally occurring pigments as food additives. As a class synthetic colorants have many advantages, such as a uniform and reproducible color, color stability, absence of flavor, and an oxidative and/or thermal and/or photostability superior to naturally occurring pigments, broad availability relatively insensitive to changes in crop yields and so forth. As a result, the popularity of synthetic colorants at least is understandable.
However, with heightened awareness of a consuming public to food additives and increased testing of some representative examples came a concern about their safety. Recent years have seen some materials formerly used as food colorants run the gamut from being beyond reproach to being suspect and even banned or at least used restrictedly. For example, FD&C Red No. 2 and FD&C Violet No. 1 have been banned in the United States and many other countries. Because of a variety of allergic reactions in sensitive individuals induced by FD&C Yellow No. 5 a recent ruling by the FDA requires food colored with it be declared as such on product labels. As a consequence the pendulum has begun to swing once more toward naturally occurring pigments as food additives.
The pigments produced by Monascus species traditionally grown on rice in the Orient are orange and relatively insoluble in water, but readily react with compounds containing amino groups to form water soluble red colorants. The water-insoluble orange pigments elaborated by Monascus have been used in the Orient for hundreds of years as a general food colorant and as a colorant for wine and bean curd, can be made water soluble or oil soluble, are stable at a pH range 2-10, are heat stable and can be autoclaved. In oriental countries Monascus microorganisms typically are grown on grains of rice and once the grains have been penetrated by the orange-red mycelium the whole mass is finely ground with the resulting powder used as a food colorant. The orange pigment is a mixture of monascorubrin and rubropunctatin, whose structures were elucidated by B. C. Fielding et al., Tetrahedron Letters No. 5, 24-7 (1960) and Kumasaki et al., Tetrahedron, 18, 1171 (1962), and which differ in the former having a 7-carbon ketonic group and the latter having a 5-carbon ketonic group. For the purposes of this application, "precursor pigment" refers to any mixture of water insoluble orange pigment containing monascorubrin and rubropunctatin as produced by fermentation of a suitable Monascus species.
Commercial production of precursor pigment requires development of a suitable fermentation procedure, which has been the subject of many reports in recent years. Shepherd et al., U.S. Pat. No. 4,145,254, made an important advance by using a two-stage process in which the microorganism first was cultivated at pH 4-7 in a growth-promoting medium, then was transferred to a second medium at pH 2-4 to stimulate precursor pigment production. The low pH did not interfere with precursor pigment production but inhibited its subsequent reaction with amino groups of proteins and/or ammonium ions in the medium. The result was the exclusive production of orange precursor pigment as a colorant. As another example U.S. Pat. No. 4,442,209 claims to increase precursor pigment formation by cultivating a Monascus species in a medium containing maltitol.
As Shepherd et al. noted, ". . . if it is desired commercially to obtain a pigment having a perfectly determined structure which may be subjected to rigorous tolerance tests and which shows perfectly reproducible properties, it is the high-yield production of a high-purity orange pigment which should be researched in the first instance." All processes described to data retain serious disadvantages associated with the separation and isolation of high purity precursor pigment. The shortcomings and difficulties inherent in the recovery of precursor pigment using prior art methods of precursor pigment production, as well as the stark contrast between our method of pigment production as embodied in the claimed invention and the prior art approach, will become apparent by a short sojourn through the prior art.
During fermentation of Monascus species the precursor pigment is produced as a metabolite strongly associated with lipids produced by the fungus. The precursor pigment is not elaborated extracellularly but remains in the mycelium as a lipid complex. Although some of the precursor pigment can be isolated by extracting the entire concentrated culture (cells plus broth) with a solvent such as ethanol at room temperature, more complete precursor pigment recovery requires that the fungal cell membranes and lipids be dissolved to release the associated precursor pigment. The majority of the pigment can only be released from the fungal cell mass by repeated extraction with hot solvents such as methanol, ethanol, isopropyl alcohol, ethyl ether or chloroform in a reflux apparatus.
Cell membrane lysis with hot solvents affords the precursor pigment-lipid complex as an ill-defined heterogeneous dispersion of liquid fatty globules. Because the fatty liquid can not be directly separated from the aqueous phase in which it is dispersed, typically one extracts the mixture with a water-insoluble organic solvent to afford as an extract a solution of the precursor pigment-lipid complex plus a myriad of other organic lipophilic components released upon cell lysis. Removal of solvent from the extract affords as a concentrate organic material generally containing substantially less than 50 percent precursor pigment, a direct consequence of strong pigment-lipid association. Crystallization of the precursor pigment from the concentrate using a suitable organic solvent then can afford, for the first time, crystalline precursor pigment of perhaps 90 percent purity, with subsequent recrystallization necessary to achieve higher purity. In other variants the precursor pigment is separated from lipids and purified by chromatography.
The foregoing extraction-chromatography-crystallization procedures are inefficient as regards yield of the purified precursor pigment. The procedures also are costly because of the use of solvents to extract the pigment and the necessity of expending energy to evaporate solvent from the extract. Such procedures also are not readily adaptable to continuous fermentation with continuous precursor pigment production.
Although there may be other aspects of precursor pigment production from Monascus which need attention, for example, obtaining suitable mutants or otherwise genetically altered microorganisms, this application is directed solely to an improvement in precursor pigment production which eliminates the need for costly extraction methods. My invention is based on several interrelated observations which serve as gross departures from the prior art. One critical observation is that in the fermentation of Monascus species the orange precursor pigment can be induced to form as a crystalline, extracellular product. The second observation is that crystalline, extracellular orange precursor pigment as formed in fermentation of Monascus species migrates to a lipophilic phase in contact with the culture medium. Especially where the lipophilic phase is chosen as to only slightly solubilize the crystalline pigment, the resultant aggregation of crystalline pigments in a water immiscible oil phase permits their facile separation merely by separating the lipophilic phase from the aqueous phase and collecting the crystals dispersed in the former. Of enormous significance is the fact that the collected crystals, after removal of adhering lipophilic phase, may be as high as 99% pure! My discovery also permits the development of both a batch recycle process for crystalline pigment production as well as a process based on continuous fermentation. In my process no toxic solvents are used which kill the cells, and the latter can be used for repeated cycles of pigment production.